Polyamines are essential growth factors for cells.1 Tumor cells have been shown to contain elevated polyamine levels and have active polyamine transport systems to import exogenous polyamines.1a This unique characteristic of cancer cells allows for cell selective drug delivery of polyamine-drug conjugates to particular cell types.1-2 The polyamine transport system (PTS) is an important target as many cancer cells need to import polyamines in order to sustain their growth rate, especially in the presence of polyamine biosynthesis inhibitors like difluoromethylornithine (DFMO).1-2 DFMO resembles the natural substrate ornithine for ornithine decarboxylase (ODC), a key enzyme in putrescine biosynthesis.1-2 
Although the PTS has been recognized as an important target for cell selective drug delivery, the PTS is still poorly understood. What is known is that the PTS is an energy-requiring and carrier mediated process.3 Recently, in mammalian polyamine transport two models of polyamine transport that have been proposed by Poulin4 and Belting5, respectively. Poulin suggested that polyamines enter the cell through an active plasma membrane transporter, followed by the sequestration into polyamine sequestering vesicles (PSV's).4a In order for polyamines to internalize within these PSV's, the process needs to be driven by a vesicular H+/polyamine carrier, which also aids in the escape from the PSV.4a It was also found that the PSV's colocalized with acidic vesicles of the late endocytic compartment and the trans Golgi.4a Belting, on the other hand, provided a multi-step endocytic process where polyamines bind to heparan sulfate proteoglycans in caveolae.5b Once the polyamines have bound to heparan sulfate, they are then endocytosed and their heparan sulfate chains are cleaved and further processing by NO liberates the polyamines.5b 
Drug conjugates, which join a cytotoxic agent to a polyamine, have exhibited selective and enhanced cytotoxicity to cancer cells compared to their normal cell counterparts.1-2 To assess whether there was any advantage in tethering a polyamine message to a toxic agent, a method was needed to investigate if these drug conjugates were indeed targeting the polyamine transport system. Delcros et. al. demonstrated that the Chinese hamster ovary (CHO) cell line was very effective for identifying drug conjugates which are PTS selective.6 This cell line was chosen along with its mutant (CHO-MG) in order to demonstrate which drug conjugates effectively utilize the PTS and which ones provide non-specific toxicity.2g, 6 The CHO-MG cell line developed by Flintoff et. al.7 is polyamine transport deficient. This mutant cell line was developed by treating the CHO cell line with ethyl methanesulfonate and subsequent dosing of the surviving cells with methylglyoxal-bis(guanylhydrazone) (MGBG) to determine their resistance to cytotoxic MGBG.7b The surviving cells that were found to be resistant to MGBG were also found to have a marked decrease (less than 1% as compared to the wild type) in the uptake of radiolabeled spermidine, demonstrating a defective PTS.7b When evaluating the polyamine drug conjugates, the IC50 is determined for both CHO and CHO-MG, with their ratio (CHO-MG IC50/CHO IC50) being utilized as an evaluator for the PTS selectivity of the compound. Compounds with high CHO-MG/CHO IC50 ratios are considered PTS selective.
Several factors can affect PTS selectivity. For example, polyamine oxidase (PAO) activity severely reduces the PTS selectivity of polyamine based drugs via drug degradation. Since polyamine oxidase targets primary amines, it also metabolizes polyamine based drugs. Since cancers have higher polyamine uptake than normal cells, polyamine-based drugs offer the opportunity to selectively target cancers via their reliance on polyamine uptake. Polyamine drugs which are stable to amine oxidases are thus needed to create anticancer agents which selectively target cancers, while having enhanced stability to amine oxidases.